Monoclonal antibodies to Sclerotinia homoeocarpa

ABSTRACT

This invention provides monoclonal antibodies which specifically bind to Sclerotinia homoeocarpa the causative agent of dollarspot disease in plants. Hybridoma producing the antibodies as well as materials and kits for carrying out the detection of Sclerotina homoeocarpa are also disclosed.

FIELD OF THE INVENTION

This invention relates to the field of diagnostic plant pathology. Morespecifically the invention relates to the immunological detection ofvarious taxa of fungi known to be the etiologic agents of a variety ofplant diseases.

BACKGROUND OF THE INVENTION

Fungi as a group cause many plant diseases. For purposes of discussionthe fungi can be classified as belonging to one of three major taxonomicclasses: Basidiomycetes, Phycomycetes, or Ascomycetes.

Basidiomycetes

Members of this class are identified by the presence of a sexual-sporeforming structure known as a basidium. Pathogenic forms include smuts,rusts and fleshy species such as mushrooms. Examples include wheat rust,white pine blister, cedar-apple rust, and smuts causing disease in corn,oats, barley, onions and wheat.

Phycomycetes

Members of this class are considered to be more primitive than membersof either the Ascomycetes or Basidiomycetes, their distinguishingmorphological feature being the absence of mycelial crosswalls. Examplesof disease caused by members of the class include the downy mildews ofgrape and other hosts, root rot and late blight of potato and tomato.

Ascomycetes

Members of this class possess a specialized reproductive structure (anascus) in which meiosis and sexual spore formation take place. Examplesof the more common plant diseases in which Ascomycetes have beenidentified as the etiologic agent include: powdery mildews on cereals,fruits and many other crops; Dutch elm disease; ergot of grains; peachand plum brown rot; black spot of roses as well as apple scab.

With respect to the present invention, members of the familySclerotiniaceae, and particularly the genus Sclerotinia, are ofparticular interest. Several hundred species have been at one time oranother assigned to this genus; many of these are important plantpathogens. The family Sclerotiniaceae was erected and discussed atlength by Whitzel (Mycologia, 37: 648-714, 1945). The taxonomy of themembers of the family, and of the genus Sclerotinia, is currently in astate of flux, with many of the members traditionally assigned to thegenus being assigned elsewhere (L. Kohn, Phytopathology, 69: 881-886,1979). However, for the purposes of the present invention, any referenceto the genus Sclerotinia will be understood by those skilled in the artto mean the genus Sclerotinia sensu lato.

As noted above, the members of the family Sclerotiniaceae contain anumber of significant pathogens. For example, several species of thegenus Monilinia Honey is known to cause brown rot of many stone fruitssuch as peach, plum and cherry, particularly in Europe. Sclerotiniasclerotiorum DBy. causes one of the most widespread diseases ofvegetable and field crops attacking clover, sunflower, various bulbousspecies, lemon, cabbage, tomato and carrot among others. The speciesSclerotinia homoeocarpa Bennett causes "dollarspot disease", awidespread and highly destructive disease of turfgrasses in Australia,North America and Europe. Again, the taxonomy of the organism issomewhat uncertain, and more than one species may be included in thenominal species. However, for present purposes, the use of the nameSclerotinia homoeocarpa is to be understood in its broadest sense, andis intended to include any causative agent of dollarspot diseases.

The above diseases are capable of causing a tremendous economic loss tothe growers of afflicted plants each year. Although various chemicaltreatment methods are known by which these diseases can be controlled tosome extent after the presence becomes evident, it is preferable to havea method of detection of the presence of the organism before it has hadan opportunity to spread very far. Therefore, a system which allows forvery early detection of the presence of the pathogenic fungus would beof tremendous value to the growers and/or keepers of potentiallyafflicted plants. The present invention enables just such a system to beput into practice by providing monoclonal antibodies which are capableof detecting the presence of Sclerotinia antigens, thus allowing earlydiagnosis of the disease, and possible prevention of widespread lossesto the affected crop.

Hybridoma Monoclonal Antibody Technology

The use of somatic hybrid cell lines as sources of antibody toindividual antigens generally dates from the work of Kohler and Milstein(Nature, 256: 495-97(1975)). The antibodies produced are quite differentthan those recovered from antiserum from conventionally immunizedanimals. Each hybrid cell line synthesizes a homogenous immunoglobulinthat represents but one of the myriad of types of antibodies that ananimal can synthesize in response to an antigen in vivo. Since eachimmunoglobulin-producing clone is characterized by the single type ofantibody it produces, the term monoclonal antibody has been adopted. Theadvantages of monoclonal antibodies are numerous; they can be obtainedin large supply; the preparation is homogenous with respect to antigenreactivity and remains so over time.

The principle of hybridoma/monoclonal technology is predicated on theobservation that when two somatic cells are fused the resultant hybriddisplays characteristics of both of the parent cell types. In the caseof monoclonal antibody production, the ability to synthesize theparticular antibody is derived from an immunocompetent cell (usually aspleen cell) taken from an immunized donor animal, whereas the abilityto continuously divide in cell culture is contributed by the otherfusion partner, a tumor cell line (often a myeloma). Early fusions werecomplicated by the fact that myeloma cell line also produced amonoclonal antibody; thus the hybrid often produced two types ofmonoclonal antibody, one of myeloma origin and the other directed by thegenetic information of the immunocompetent cell. Subsequently, tumorcells lines incapable of producing their own monoclonal have been used,e.g., SP2/0-Ag14 or X63-Ag8.653, thereby simplifying the analysis of theresultant fusion products.

Another technical consideration involves the rationale for selecting thesuccessful fusion events (hybrid cells) from the two types of parentalcells. Routinely a million or more cells of each type are used in thefusion protocol, and since fusion does not occur with 100% frequency,the job of trying to recover fusion products from the high background ofunfused or self-fused parents can be formidable. As mentioned hybridomasare formed by the fusion of short-lived antibody producing (spleen)cells and long-lived myeloma cells. The desired result is a long-livedcell line which produces antibody. Since the spleen cells have a finitelife span in culture one can simply wait an appropriate period for allthe nonfused or self-fused spleen cells to die; however one must stillrecover from the resultant population the long-lived antibody producingcells from the long-lived antibody non-producing cells. A popular meansfor selection hybrid cells is the so-called HAT-selection system. Thissystem involves the use of the enzymehypoxanthine-guanine-phosphoribosyl transferase (HGPRT). This enzymefunctions in the purine salvage pathway in mammalian cells. These cellsare also capable of synthesizing purines de novo. Under most conditions,both pathways probably operate to a certain extent. If a cell lacksHGPRT, the salvage pathway is blocked and purines must be manufacturedfrom non-purine materials.

The chemical 8-azaguanine is an antimetabolite which is capable ofmasquerading as the purine guanine and replacing it in some of itsnormal reactions. Azaguanine is incorporated into DNA, interfering withthe normal growth pattern and leading to cell death. Since azaguaninemust be salvaged, any cell which lacks HGPRT activity cannot utilizeazaguanine and will grow in its presence.

A selective system which operates on the same enzyme but in the oppositesense in that HGPRT positive cells are selected is described by J. W.Littlefield (Science, 145: 709 (1964)). It is called HAT and containshypoxanthine, aminopterin and thymidine (HAT medium). Aminopterin is anantimetabolite that prevents de novo purine synthesis and methylation ofdeoxyuridylate to form thymidylate. Hypoxanthine can serve as asalvagable purine in the event that aminopterin blocks de novo purinebiosynthesis while thymidine bypasses the necessity for the methylationof thymidylate. Thus, in the presence of aminopterin, any cell withpositive HGPRT activity will proliferate while cells with negative HGPRTactivity will die.

In the hybrid system used for selection in accordance with thisinvention, the myeloma cells are resistant to azaguanine and susceptibleto aminopterin, that is, they are HGPRT negative. Thus, they will die inthe presence of aminopterin. The antibody producing cells are HGPRTpositive. By fusing the cells and growing them in HAT medium withoutazaguanine (HT medium), the successfully fused cells are selectedbecause the myeloma cells which constitute the proliferating line canonly grow where HGPRT activity is present and this activity must besupplied by the HGPRT positive cell line. The antibody producing HGPRTpositive cell line are not killed in this medium. They will live for atime but will not proliferate.

Thus, by fusing the cells in a HAT medium, systems are produced in whichthe myeloma cells and antibody producing cells can grow long enough toproduce hybrid cells but in which only the hybrid cells can survive andproliferate. After selection each hybridoma clone is then screened forthe ability to produce the particular antibody of interest.

The hybridoma/monoclonal antibody technology has been tremendouslysuccessful, one indication being the dedication of nine entiresub-classes within United States Patent Trademrrk Offices classificationsystem to this technology (935/100 et seq.). Illustrative of theactivity is the field of monoclonal antibody technology are U.S. Pat.No. 4,196,265 relating methods of producing monoclonal antibodies toviruses; U.S. Pat. No. 4,404,279 relating to methods of culturinghybridomas and increasing hybridization and U.S. Pat. No. 4,427,653relating to a method of making monoclonal antibodies in which theantigen preparation is pre-absorbed with certain monoclonal antibodiesprior to immunization. Although by no means an exhaustive list,monoclonal antibodies have been developed to the following antigens:Treponema pallidum (EPO-83302898.8), hepatitis antigens(EPO-83103858.3), anti-H-Y. (EPO-83301214.9), lens epithelial cells(83301176.0), carcinoembryonic antigen (PCT-W081101469), urokinase(EPO-83100190.4), herpes (EPO-83400074.7), rat hepatocyte (82306409.2),Schistosoma mansoni (PCT-W083/01837), Leishmania (PCT-W083/01785,transferrin receptor glycoprotein (EPO-82305658.5), rheumetoid factor(PCT-W083/01118) cell surface antigens of human renal cancer(EPO-82107355.8) alphainterferon (PCT-W081/02899), T-cell antigen(EPO-81300047.8) human suppressor T-cells (EPO-80304348.8).

With respect to plant diseases, Hsu, H. T, et al. (ASM News, 50(3):99-101 (1984)) list 18 plant virus species to which monoclonalantibodies have been developed; included are carnation etched ringvirus, potato leaf roll virus, southern bean mosaic virus, tobaccomosaic virus, tomato ringspot virus, and tulip breaking virus.

Monoclonal antibodies to fungal organisms have been developed primarilyas a tol for human disease diagnosis. For example, U.K. PatentApplications GB2138444A and GB2138445A relate to monoclonal antibodiesreactive with Candida and Aspergillus, respectively.

Disclosed herein are monoclonal antibodies specifically reactive withmembers of the fungal family Sclerotiniaceae and methods for theirproduction. The antibody is particularly useful for broad rangedetection of Sclerotinia infections.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a hybridoma which produces a monoclonalantibody to an antigen from at least one strain of Sclerotinia.

In a further embodiment the invention provides a monoclonal antibody toan antigen of at least one strain of Sclerotinia.

In a further embodiment the ineention provides a method for preparinghybridomas capable of producing monoclonal antibody to an antigen of afungus belonging to the genus Sclerotinia comprising:

providing a crude extract of Sclerotinia antigen; immunizing an animalwith said extract;

recovering immunocompetent cells from said animal;

fusing said immunocompetent cells with myeloma cells to form hybridomas;

detecting those hybridomas capable of producing monoclonal antibodies toSclerotinia antigens by affixing the antigen to be detected to a solidsupport by means of glutaraldehyde cross-linking;

and indicating the presence of monoclonal antibody complexed with saidaffixed antigen by means of an avidin-biotin enzyme-linked immunoassay.

In a further embodiment the invention provides a method for detectingthe presence of Sclerotinia antigen in a sample containing samecomprising:

forming a binary complex between said antigen and a first antibodycapable of reacting with said antigen;

forming a tertiary complex by contacting the binary complex with asecond monoclonal antibody;

detecting the presence of said tertiary complex by contacting thetertiary complex with an analytically detectable immunological reagent,thereby detecting the presence of said Sclerotinia antigen.

In a final embodiment the invention provides a kit for the immunologicaldiagnosis of Sclerotinia infection of plants comprising a carrier beingcompartmented to receive in close confinement therein:

an antigen extraction means;

a solid support having affixed thereto a antibody capable of forming abinary complex with Sclerotinia antigen; and

a binary complex detecting means.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods for the production of monoclonalantibodies to Sclerotinia homoeocarpa, the monoclonal antibodies per se,the hybridoma cell line capable of producing said antibodies and methodsand kits employing the monoclonal antibodies to diagnose Sclerotiniainfection in plant tissue.

Method of Extraction of Fungal Proteins

Fungi were cultured in 50 ml of PDB (Potato Dextrose Broth) medium in250 ml flasks, (2 liters of Sclerotinaa homoeocarpa Fitz. were generallyemployed). After one week the fungal cultures were harvested from themedium, washed twice in PBS (Phosphate buffered saline; pH 7.4). Fungalcultures were transferred into a 300 ml batch chamber of a DYNO-MIlltype KDL containing 240 ml of 0.50 mm/lead-free glass beads [IMPANDEX].Cooling jacket of the Batch chamber was pre-cooled to 8° C. with coldtap water. Extract was ground at 3000 RMP for 5 minutes after which thecontents of the batch chamber were transferred to 50 ml polystyrenetubes and centrifuged at 17,000 RPM (34,540 g) in a Sorvall RC-5Brefrigerated centrifuge using a size SS-34 rotor. The fungal supernatantwas aliquoted and frozen until use. Total protein content of sampleswere in the range of 0.5-2 mg/ml.

Monoclonal Antibody Production

The procedure is a modification of that developed by Kohler and Milstein(Nature, 256: 495 (1975) and Hammerling (Eur. J. Immunol., 7: 743(1977)).

INJECTIONS: The test animals were 4-5 weeks old female

BALB/c mice purchased from CHARLES RIVER BREEDING

LABORATORIES, INC., Wilmington, Mass.

Day 1

1st injection: 0.05 mg of fungal protein in 0.1 ml of PBS buffer plus0.1 ml Freund's complete adjuvant. IP injection

Day 22

2nd injection: same as above

Day 36

3rd injection: 0.025 mg fungal protein in 0.05 ml solution in 0.05 mlFreund's complete adjuvant. IP injection

Day 38

Fusion

Spleen isolation

Each animal was sacrificed by cervical dislocation. The spleen wasremoved and placed in 20 ml of Dulbecco's Modified Eagle's Medium. Thespleen was placed on an 80 mesh sterile screen. The spleen was then cut,perfused with DMEM (Dulbecco's Modified Eagle Medium cat no. 320-1965Gibco Labs.) and then gently massaged with a sterile plunger from a 0 ccdisposable plastic syringe. During the entire process of spleen cellextraction, the screen was continually rinsed with DMEM. Contents werepipetted into a 50 ml disposable centrifuge tube and spun down at 1200RPM for 10 minutes (centrifugation done at room temperature). Thesupernatant was decanted and the cell pellet washed with 10 mls of redblood cell lysing solution (0.83% NH4Cl; 0.01M KHC03; 0.lmM EDTA) for 90seconds at room temperature. The lysing reaction was stopped by dilutingwith 40 mls of DMEM. The sample was left to stand for 3 minutes, and thesupernatant pipetted to 50 ml centrifuge tubes. After centrifugation,the pellet was washed with 50 ml of DMEM and recentrifuged. The finalpellet was resuspended with 5 ml of DMEM. A small sample of the spleencells was retained for counting and to check for cell viability. Optimalconcentration of spleen cells is 10 to the 7 cells per ml.

Myeloma cells (SP2-0-Ag 14) obtained from American Type CultureCollection) were transferred (concentration 1× 10⁶ cells per ml) fromculture into a 50 ml Falcon tube. The myeloma cells for fusion werecentrifuged (1200 RPM for 10 minutes at room temperature). Aftercentrifugation, the supernatant was discarded into a clean glass beaker,the cells were washed with DMEM, and recentrifuged. The spleen cellswere added to the tube containing the washed myeloma pellet. The myelomaand speen cells were gently resuspended with the aid of a 10 ml pipetteand automatic pipetter and centrifuged for 10 minutes at 1200 RPM atroom temperature. Following centrifugation, the supernatant wasdecanted.

Fusion

The fusion medium, 50% PEG (polyethylene glycol) 1500 (M.A. BioproductsCat. #17-7802) prewarmed to 47° C., was suspended in DMEM. One ml offusion medium was added dropwise to the tube containing the resuspendedmyeloma and spleen cells--time thirty seconds. The final 7 minutes ofthe fusion reaction was to allow the gradual dilution of the PEG withDMEM. At the end of the dilution, the final volume in the tube reached50 mls. During the entire fusion period, the tube was gently tapped toinsure proper mixing of the material. The tube was then centrifuged(1200 RPM for 10 minutes at room temperature) and the supernatatremoved. Prewarmed HAT medium (described below) (33 ml) was added to thetube, and the cellular contents were suspended using a 10 ml pipette.The final concentration of spleen cells was 1.4×10⁶ cells.

Cells were then added to the 60 central wells of a 96 well microtiterplate (Limbro multiwell). To each well was added 150 ul of fusedMyeloma/Spleen material. Outer wells of the microtiter plate were thenfilled with HAT medium. Microtiter plates were placed in a waterjacketed 7% CO₂ incubator, temperature 37° C.

Cells were refed with HAT medium every 4 days. Visible hybridoma growthbegan to appear after 7 to 10 days. A number of different hybridomalines were produced by the foregoing procedure, each of which producesmonoclonal antibodies of the IgG class. The preferred hybridoma isdesignated as SH3LLIBll-G6-D3. The antibody produced by this hybridomais of the IgGl subclass.

    ______________________________________                                        HAT Medium Composition                                                        ______________________________________                                        DULBECCO'S MODIFIED EAGLE MEDIUM                                                                        766    ml                                           cat # 320-1965 GIBCO LABS                                                     L Glutamine (200 mM) 100 × concentration                                                          10     ml                                           cat # 320-5030 GIBCO LABS                                                     Pencillin/Streptomycin solution:                                                                        10     ml                                           10,000 μ/ml            10     mg/ml                                        cat # P0781 SIGMA                                                             Aminopterin (50 ×)  4      ml                                           cat # A-5159 SIGMA                                                            Hypoxanthine/Thymidine solution:                                                                        10     ml                                           Thymidine                                                                     cat # T-9250 SIGMA        38.8   mg                                           Hypoxanthine              136.1  mg                                           cat # H-9377 SIGMA                                                            add 100 ml sterile water and pH to 8.5 with                                   sterile 1 N NaOH                                                              Fetal Bovine Serum        200    ml                                           cat # 12-10378 HAZLETON DUTCHLAND,                                            INC.                                                                          ______________________________________                                    

Screening for Hybridomas

Those hybridomas producing antibodies to fungal pathogens wereidentified by using prepared Sclerotinia homoeocarpa (Eds.) Fitz. fungalmaterial (protein concentration 10 ug/ml in PBS buffer) and materialfrom related species in an avidin/biotin amplified glutaraldehyde ELISAformat.

Standard Screening Protocol

This procedure relates to an enhancement procedure for screeninghybridomas secreting antibodies to fungal pathogens.

AVIDIN/BIOTIN GLUTARALDEHYDE

ELISA SCREENING

ELISA - GLUTARALDEHYDE Procedure

1. 200 μl of glutaraldehyde buffer was placed into each well (Immulon Iplates), incubated for 3 hours at 55° C., cooled to room temperature andthe plates washed 3 times with deionized (DI) water.

2. 200 μl of antigen diluted in 0.15M PBS, pH 7.2, was dispensed intoeach well. One row was left empty for use as the glutaraldehyde control.The mixture was incubated for 24 hours at 4° C., the remainingsuspension discarded and washed 3× with PBS.

3. 200 μl of (mono)ethanolamine solution was dispersed into each well,incubated for 20 hours at 4° C., the remaining solution discarded andplate washed 3X with PBS.

4. 200 μl of appropriate sample was placed into each well, incubated for2 hours at 33° C. with humidity. The remaining solution was discardedand the plate washed 3× with PBS.

5. The supernatants were aspirated and washed 2 times with 200 μl PBS.

6. Biotinylated anti-mouse IgG or IgM; peroxidase conjugated avidinreagent (VECTOR LABORATORIES mouse anti IgG or IgM; ABC reagent)

10 ml PBS+100 μl normal horse serum+1 drop biotinylated anti-mouse IgG

10 ml PBS (0.1% tween)

add 2 drops ABC reagent A

immediately add 2 drops ABC reagent B, mix and let

stand for 30 minutes before using

7. 75 μl/well biotin/anti-mouse solution was added and incubated for 30minutes at room temperature.

8. The mixture was aspirated and washed 2 times with 200 μl PBS.

9. ABC reagent (see above) was added at 75 μl well incubated 30 minutesat room temperature, then aspirated and washed 5 times with 200 μlPBS/well.

10. The following substrate solution was added at 200 μl/well.

Citrate Phosphate Buffer

7.1 g Na₂ HPO₄ (500 ml)

9.6 g citric acid (500 ml)

adjust pH of first solution to 6.0 by adding citric

acid

50 ml buffer

20 mg Phenylenediamine-HCL 1,2 benzenediamine (OPD)

Sigma P 3888

167 μl 3% H₂ O₂

The mixture was incubated at room temperature for 10 minutes andabsorbance read at 405 nm. Table I shows results observed with anascites reagent produced with a single cell line, SH3LLIBll-G6-D3,diluted 1:500 in 20% Fetal Calf Serum (FCS) in Dulbecco's ModifiedEagle's Medium (DMEM).

Required Solutions

1. Glutaraldehyde buffer: 0.1% glutaraldehyde in 0.1M carbonate buffer.The carbonate buffer, pH 9.0, consists of 1.57 g Na2CO₃ and 2.93g NaHCO₃per liter of DI water.

2. PBS-tween: 8.0 NaCl, 0.2g KH₂ PO₄, 2.9 g, 1.15 g Na₂ HPO₄ anhydrous,0.2 g KCl, per liter of DI water, pH 7.4.

3. (Mono)ethanolamine solution: 1 mg/ml solution (1 g/liter of DIwater).

                  TABLE I                                                         ______________________________________                                        Test With SH3LLIB11-G6-D3                                                     Fungal Cultures                                                                          Source             Absorbance                                      ______________________________________                                        Sclerotinia homoeocarpa                                                       SH-1       Cole; Penn State   1.05   3+                                       SH-2       Cole; Penn State   0.47   2+                                       SH-3       Cole; Penn State   0.55   2+                                       SH-4       Wilkinson; Illinois                                                                              0.18   1+                                       SH-5       Gail Worf; Madison, WI                                                                           0.29   2+                                       SH-6       Gail Worf; Madison, WI                                                                           0.10   +/-                                      SH-7       Gail Worf; Madison, WI                                                                           0.43   2+                                       SH-8       Scotts; Marysville, OH                                                                           0.68   2+                                       Sclerotinia trifoliorum                                                       ST-1       ATCC #34326        0.04   --                                       Sclerotinia minor                                                             SM-2       ATCC #44236        0.03   --                                       Sclerotinia sclerotiorum                                                      SS-1       Nelson; N. Dakota State                                                                          0.04   --                                       SS-2       Nelson; N. Dakota State                                                                          0.04   --                                       SS-3       Nelson; N. Dakota State                                                                          0.04   --                                       SS-4       Nelson; N. Dakota State                                                                          0.04   --                                       SS-6       Maxwell; U. of Wisconsin                                                                         0.06   --                                       Whetzelinia (Sclerotinia) sclerotiorum                                        SS-7       Miller; Rancocas, NJ                                                                             0.03   --                                       Rhizoctonia solani                                                            RS-1       Lucas; North Carolina                                                                            0.04   --                                       RS-2       Cole; Penn State   0.07   --                                       RS-3       Cole; Penn State   0.05   --                                       RS-5       Lucas; North Carolina                                                                            0.05   --                                       RS-6       Larsen; Minnesota  0.06   --                                       RS-7       O'Neil; Beltsville, MD                                                                           0.05   --                                       RS-8       Burpee; U. Guelph, Canada                                                                        0.06   --                                       RS-10      Burpee; U. Guelph, Canada                                                                        0.08   --                                       RS-11      Burpee; U. Guelph, Canada                                                                        0.05   --                                       RS-12      Burpee; U. Guelph, Canada                                                                        0.05   --                                       RS-13      Burpee; U. Guelph, Canada                                                                        0.09   --                                       RS-14      Burpee; U. Guelph, Canada                                                                        0.08   --                                       RS-15      Burpee; U. Guelph, Canada                                                                        0.08   --                                       Pythium aphanidermata                                                         PA-1       Larsen; Schmitthener-                                                                            0.07   --                                                  Wooster, OH                                                        ______________________________________                                         adjusted protein concentration 10 μg/100 μl                        

Second Screening Procedure

In addition to the standard screening procedure described above, asecond screening was performed utilizing infected material.Specifically, a multiwell ELISA test for detection of dollarspot,Sclerotinia homoeocarpa, was performed on infected turfgrass. The testcompares results observed with both ascites and supernatants of thefollowing cell lines: SH3LLIBll-G6-D3 and SH3LLIBll-G6-B7.

The turfgrass sample extraction buffer used in the following procedureincorporates a mechanism for the selective deactivation of nativeturfgrass peroxidase enzyme, thus making it possible to use a conjugatesystem employing peroxidase.

The following components were employed in the screening procedure:

1. A 96 well plate (immulon I) precoated with glutaraldehyde asdescribed in the first screening.

2. Extraction buffer: Phosphate buffered saline (PBS, pH 7.4) with 5%polyvinylpyrrolidone (mw 14,000) and 3% hydrogen peroxide.

3. Monoclonal antibody: Ascites diluted in 20% FCS in DMEM, 1:500,1:1000.

4. Conjugate: Biotinylated goat anti-mouse IgG with peroxidaseconjugated avidin (ABC Test from Vector Labs).

5. Substrate: 1 mg/ml of urea peroxide in 0.1M sodium citrate, pH 4.5.To each 15 ml of urea peroxide solution is added 6 mg oforthophenyldiamine (OPD).

The following procedure is followed:

1. Glutaraldehyde coated plates are washed 8 times with water.

2. 100 μl of sample extract is placed in the test wells, and the plateis incubated for 2 hours at 37° C.; this is followed by washing theplate 8 times with PBS buffer.

3. 100 μl of the chosen monoclonal reagent is then placed in the testwells, and again incubated for 2 hours at 37° C., and washed 8 timeswith PBS buffer.

4. 75 μl of biotinylated goat anti-mouse IgG is further added to thewells and incubated for 30 minutes at room temperature, followed by 8washings with PBS buffer.

5. 75 μl of peroxidase conjugated avidin is then added, and againincubated for 30 minutes at room temperature, followed by 8 washingswith PBS buffer.

6. 200 μl of substrate is added to the wells and incubated for 30minutes at room temperature.

7. Samples are read at 410 nm absorbance; the results are shown in TableII.

                                      TABLE II                                    __________________________________________________________________________    Absorbance 410 nm (20 minute incubation)                                                Fylking grass                                                                        Fylking grass                                                                        Fylking grass                                                   infected with                                                                        infected with                                                                        infected with                                                                        Fylking grass                                                                        Ext.                                              SH-1   PA-1   RS-16* uninfected                                                                           buffer                                  __________________________________________________________________________    SH3LLIB11-G6-D3                                                               ascites                                                                       dil (1:500)                                                                             0.73   0.16   0.21   0.16   0.12                                    dil (1:1000)                                                                            0.81   0.14   0.14   0.09   0.06                                    SH3LLIB11-G6-B7                                                               ascites                                                                       dil (1:500)                                                                             0.56   0.14   0.17   0.09   0.05                                    dil (1:1000)                                                                            0.46   0.11   0.14   0.09   0.07                                    SH3LLIB11-G6-D3                                                               supernatants                                                                  100 ul    0.52   0.10   0.10   0.05   0.06                                    SH3LLIB11-G6-B7                                                               supernatants                                                                  100 ul    0.53   0.13   0.10   0.08   0.07                                    __________________________________________________________________________     ascites diluted in 20% Fetal Calf Serum in DMEM                               *Rhizoctonia solani Source: Burpee; U. of Guelph, Canada.                

Subcloning Procedure

Those wells giving positive responses to the ELISA tests undergo alimiting dilution so that pure strains of hybridoma cells n:ight begrown. The limiting dilution method involved culturing serially dilutedsuspensions of hybridomas. Each dilution series was set up in 6-12 wellsof a 96 well culture plate. These wells were then retested for specificantibody activity to fungal proteins. Positive wells were thentransferred to 20 ml culture flasks for mass culturing.

Deposit of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following hybridomas weremade with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. on Nov. 14, 1985. The accession number indicated wasassigned after successful viability testing, and the requisite fees werepaid. Access to said culture will be available during pendency of thepatent application to one determined by the Commissioner to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. All restriction onavailability of said culture to the public will be irrevocably removedupon the granting of a patent based upon the application and saidculture will remain permanently available for a term of at least fiveyears after the most recent request for the furnishing of a sample andin any case for a period of at least 30 years after the date of thedeposit. Should the culture become. nonviable or be inadvertentlydestroyed, it will be replaced with a viable culture(s) of the sametaxonomic description.

    ______________________________________                                        Hybridoma           ATCC No.                                                  ______________________________________                                        Balbc/SP2 SH3LLIB11-G6-D3                                                                         8944                                                      ______________________________________                                    

Detection of FungalPathogens and Kits Therefor

This invention contemplates the use of the monoclonal antibodiesdescribed above in a system for detection of Sclerotinia infection.Accordingly, a sample of plant material suspected of harboring theorganism is subjected to an extraction procedure whereby the plantmaterial is physically disrupted such as by grinding and the resultantcrude extract is diluted into water or buffer. A sample of the crudeextract is contacted with a first antibody specifically reactive with anantigenic determinant of the organism to be detected. Preferably theantibody is immobilized on a solid support such as the walls of amicrotiter plate. The antibody may be a monoclonal antibody or acomponent of polyclonal sera. After removing the unreacted material bywashing, the resulting binary complex (antigen-antibody complex) iscontacted with a monoclonal antibody specifically reactive to theantigen to be detected. Of course if a monoclonal is employed as thefirst antibody the second monoclonal should be reactive with a differentantigenic determinant than the first monoclonal, unless it can be shownthat the determinant is present in multiple copies in the antigen. Bycontacting the immobilized binary complex with the second monoclonalantibody, a tertiary complex is formed. After washing to remove any ofsecond antibody which did not bind to the binary complex, the tertiarycomplex may be detected by a variety of analytical techniques. Thesecond monoclonal could be labelled directly and the tertiary complexindicated. Alternatively, the ELISA system described above may beemployed whereby the tertrary complex is reacted with anbiotin-labelled, antiimmunoglobulin and that reaction product issubsequently contacted with an avidin-enzyme reagent. Once reacted, thesubstrate of the enzyme is added and the enzyme reaction productdetected, thus indicating the presence of the organism or antigentherefrom.

To facilitate the detection the various reactants are provided in theform of a kit.

What is claimed is:
 1. A monoclonal antibody capable of specificallybinding with an antigen from Sclerotinia homoeocarpa.
 2. The antibody ofclaim 1 wherein the antibody is of the IgG class.
 3. A hybridoma whichproduces a monoclonal antibody which is capable of specifically bindingwith an antigen from Sclerotinia homoeocarpa.
 4. The hydridoma of claim3 wherein said monoclonal antibody is of the IgG class.
 5. The hybridomaof claim 4 wherein said monoclonal antibody is of the IgGl class.
 6. Thehybridoma of claim 5 having all of the identifying characteristics ofATCC HB
 8944. 7. A monoclonal antibody produced by culturing thehybridoma of claim
 3. 8. A monoclonal antibody produced by culturing thehybridoma of claim
 4. 9. A monoclonal antibody produced by culturing thehybridoma of claim
 5. 10. A monoclonal antibody produced by thehybridoma of claim
 6. 11. The monoclonal antibody of claim 10 whereinsaid antibody is labelled with an analytically detectable reagent. 12.The monoclonal antibody of claim 11 wherein said reagent is selectedfrom the group consisting of biotin, a fluorensent dye, a radioactivematerial and an enzyme.
 13. The monoclonal antibody of claim 10 whereinsaid antibody is immobilized onto a solid support.
 14. A method ordetecting the presence of an antigen from Sclerotinia homoeocarpa in asample containing the antigen comprising:forming a binary complexbetween said antigen and a first antibody capable of reacting with saidantigen; forming a tertiary complex by contacting the binary complexwith a second monoclonal antibody; detecting the presence of saidtertiary complex by contacting the tertiary complex with an analyticallydetectable immunological reagent thereby detecting the presence of theantigen from Sclerotinia homoeocarpa.
 15. The method according to claim14 wherein said first antibody is monoclonal or polyclonal.
 16. Themethod according to claim 15 wherein said first antibody is immobilizedto a solid support.
 17. The method according to claim 24 wherein saidanalytically detectable immunological reagent is a third antibodyreactive to said second monoclonal antibody.
 18. The method according toclaim 17 wherein said detection is by means of a label selected from thegroup consisting of biotin, a radioactive material, fluorescent dyes,and an enzyme.
 19. The method according to claim 18 wherein the label isbiotin and its presence is indicated by reaction with avidin.
 20. Themethod according to claim 19 wherein said avidin is complexed with anenzyme and said complex is detected by measuring of the enzyme reactionproduct.
 21. A kit for the immunological diagnosis of Sclerotiniahomoeocarpa infection of plants comprising a carrier being compartmentedto receive in close confinement thereinan antigen extraction means; asolid support having affixed thereto a monoclonal antibody capable ofspecifically binding with Sclerotinia antigen; and a binary complexdetecting mean.
 22. The kit according to claim 21 wherein said binarycomplex detecting means comprises a biotin-avidin enzyme linkedimmunoassay system.